Bacilli have a very long history of use in industrial large scale fermentation because of some very advantageous properties. They are capable of growing on rather cheap nutrients, they do not produce endotoxins so that they are harmless for man, plants and animals, and they are capable of synthesizing many industrially interesting proteins, such as enzymes, as extracellular products which in most cases can be isolated rather easily. This combination of characteristics, in particular the secretion of products into the culture medium, makes the use of Bacilli for commercial purposes much more attractive than for example the use of E. coli. In order to improve the economic value of Bacilli even further, many attempts have been made to develop a Bacillus host-vector system, both for homologous and heterologous gene expression. See, for example, D. Dubnau in "Experimental Manipulation of Gene Expression" Academic Press (1983) 33-51 and R. H. Doi, Biotechnology and Genetic Engineering (1984) 2:126-155.
Unfortunately, however, the level of expression and secretion appeared to be relatively low. See, for example, S. Kovacevic et al., J. Bacteriol. (1985) 162:521-528, C. W. Saunders et al., J. Bacteriol. (1984) 157:718-726, R. Ohmura et al., J. Biochem. (1984) 95:87-93, Book of Abstracts of the 3rd International Conference on Genetics and Biotechnology of Bacilli, Stanford U.S.A. (1984), K. Lunstroom, FEMS Letters (1984) 23:65-70, I. Palva et al., Gene (1983) 22:229-235, K. Lundstrom et al., Virus Res. (1985) 2:69-83, K. Hardy et al., Nature (1981) 293:481-483, K. Mosbach et al., Nature (1983) 302:543-545, S. Chang et al., NSC Ser. Gene (1981) 16:199-216 and J. I. Flock et al., Mol. Gen. Genet. (1984) 195:246-251.
Many improvements of the Bacillus cloning system have already been reported with respect to increased expression to an economically acceptable level. They are mainly related to improvements of the promoter region in order to increase the transcription efficiency (D. M. Williams et al., J. Bacteriol. (1981) 146:1162-1165, R. G. Schoner et al., Gene (1983) 22:47-57, D. S. Goldfarb et al., Nature (1981) 293:309-311, L. Band et al., Gene (1983) 26:313-315, C. E. Donelly and A. L. Sonnenshein, J. Bacteriol. (1984) 157:965-967 and Dutch patent application Ser. No. 86/201951.0), but they also concern improvements of the Shine-Dalgarno region in order to increase the translation efficiency (A. Hui et al., EMBO Journal (1984) 3:623-629, H. de Boer et al., DNA (1983) 2:231-235, L. Band and J. Henner, Biochem. Soc. Symp. (1984) 48:233-245 and European patent applciation no. 86/201951.0). The construction and use of vectors that permit isolation, improvements and use of secretory signal sequences, however, have not been reported so far.
Most of the current understanding of protein secretion in prokaryotic cells is limited to E. coli, (L. L. Randall and S. T. S. Hardy, Microbiol. Reviews (1984) 48:290-298 and S. A. Benson et al., Ann. Rev. Biochem. (1985) 54:101-134). In E. coli, a gram-negative organism, the secretion of a protein is in fact the transport to the periplasm rather than to the culture medium as is the case in Bacillus, a gram-positive organism. The knowledge of protein secretion in E. coli has mainly been obtained by the study of fusion proteins containing a segment of a protein which is known to be translocated to the periplasm, attached to .beta.-galactosidase.
A very serious limitation in the use of such hybrid proteins is that the .beta.-galactosidase moiety appears to be unable to pass through the E. coli cytoplasmic membrane, cf. C. S. Hoffman and A. Wright, Proc. Natl. Acad. Sci. U.S.A. (1985) 82:5107-5111. Although this problem has been overcome by the use of alkaline phosphatase instead of .beta.-galactosidase (C. Monoil and J. Beckwith, Proc. Natl. Acad. Sci. U.S.A. (1985) 82:8129-8133), the authors describe their results, obtained with some hybrid alkaline phosphatases as transport from the cytoplasm into the periplasm rather than as secretion into the culture medium.